Amongst imaging technologies PET (Positron Emission Tomography) plays a very important role due to its outstanding potential to visualize physiological processes at the molecular level in real time. PET is therefore essential in clinical diagnostics and has gained major significance in drug development. Beside technical improvements, PET benefits mainly from innovations in the field of tracer development, comprising both progress in labelling strategies and an intelligent design of selective molecular probes with the capability to visualize molecular targets involved in physiological and pathophysiological processes. A prerequisite for the latter is a deepened insight into the biology underlying normal or diseased states at the molecular level. Molecular probes for PET-imaging must be labeled with suitable γ+-emitting nuclides. Among the spectrum of easy available radionuclides 18F-fluorine is still the nuclide with the highest impact in PET research. This is mainly based on the excellent nuclear properties of 18F in comparison to other cyclotron-produced nuclides. Decay characteristics of 18F [E(β+)=630 keV, abundance: 97%; t1/2=109.8 min] make it an ideal PET-isotope with respect to half-life and resolution.
Numerous methods for 18F labelling have been developed to prepare tailor-made probes which allow visualizing biochemical processes of interest. The vast majority of 18F-labelling techniques are based on aliphatic and aromatic nucleophilic substitution reaction with 18F−. Sometimes, 18F-labeled small molecules can be obtained in one step from the proper labelling precursor. However, protecting groups are often required for functionalities in the molecule which may interfere with the radiofluorination reaction.
Relatively harsh reaction conditions for radiofluorination are normally incompatible with sensitive molecules including proteins and the majority of peptides. In this case indirect radiofluorination via 18F-labeled prosthetic groups is the only alternative. Radiofluorinated active esters—amine-reactive prosthetic groups—are among the most widely used radiolabeled building blocks.
In all previously described radiosyntheses of 18F-labelled active esters [18F]fluoride should be preliminary taken up in an aqueous or aqueous/organic solution of moderately strong or weak bases to give the corresponding [18F]fluoride salt. Usually K, Cs or tetraalkylammonium carbonates/bicarbonates are used. In case of K salts aminopolyethers are routinely added to enhance the nucleophilicity of 18F−. In case of Cs and tetraalkylammonium salts enhancement of 18F− nucleophilicity is achieved as a result of charge separation based on the great difference between the sizes of counter ions. However, active esters are limitedly stable under basic conditions. Consequently, a majority of them including N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) could not be usually prepared in one step in acceptable RCYs. Only few of them such as 6-[18F]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester ([18F]F-Py-TFP) could be prepared via direct radiofluorination. Furthermore, water substantially diminishes nucleophilicity of 18F− due to tenacious hydration. Consequently, water removal using repetitive azeotropic drying with acetonitrile is usually mandatory.
[18F]SFB is most commonly used for the preparation of radiolabelled biomolecules. However, its broad application is hampered by tedious preparation procedures. Some of them are summarized in Table 1.
TABLE 1Selected methods for the preparation of [18F]SFB.StepsPrecursorRProcessTime [min]RCY [%]Ref3H     OEt3-step, 3-pot, 3 separations (SPE & HPLC) 3-step, 2-pot, 2 separations (SPE) 3-step, 1-pot, 1 separation (SPE)80     78   6030-35     41-51   441     2   3 OtBu3-step, 2-pot, 26834-384separations (SPE) 2—2-step, 2-pot, 2 separations (SPEs or SPE & HPLC)100 (for 2 SPE); 160 (for SPE & HPLC)30-405G. Vaidyanathan, M. R. Zalutsky, Nucl. Med. Biol. 1992, 19, 275-281.; H. -J. Wester, K. Hamacher, G. Stocklin, Nucl. Med. Biol. 1996, 23, 365-372.; S. Guhlke, H. H. Coenen, G. Stöcklin, Appl. Radiat Isot. 1994, 45, 715-725.; E. D. Hostetler, W. B. Edwards, C. J. Anderson, M. J. Welch, J. Label. Compd. Radiopharm. 1999, 42, S720-S722.; M. Glaser, E. Årstad, S. K. Luthra, E. G. Robins, J. Label Compd. Radiopharm. 2009, 52, 327-330.
All described [18F]SFB radiosyntheses consist of 2-3 reactions and multiple operation steps. For example, according to Wester et al. (Nucl. Med. Biol. 1996, 23, 365-372) [18F]SFB was prepared via ethyl 4-[18F]fluorobenzoate (Scheme 1).

Accordingly, 18F− target water was added to a solution of K2CO3/K2C2O4 and K2.2.2 (Kryptofix 222) in aqueous MeCN. The solvent was removed under reduced pressure in a flow of nitrogen and the residue was two times azeotropically dried by using MeCN. The residue was taken up in a solution of N,N,N-trimethyl-4-carbomethoxyanilinium triflate in DMSO. The reaction mixture was heated at 90-110° C. for 6 min. Thereafter, 1 M NaOH was added and the mixture was heated at the same temperature for a further 10 min.
Afterwards, it was acidified with 1 M HCl and diluted with water. The intermediate 4-[18F]fluorobenzoic acid was purified by SPE (solid phase extraction) using polystyrene and cation exchange cartridges. Tetramethylammonium hydroxide was then added to the methanolic solution of the intermediate and MeOH was removed under reduced pressure at 90° C. The residue was two times azeotropically dried by using MeCN. To the residue a solution of TSTA (N,N,N′,N′-tetramethyl(succinimido)uronium tetrafluoroborate) in MeCN was added and the mixture was heated at 90° C. for 2 min. [18F]SFB was finally isolated by SPE using a polystyrene resin.

Glaser et al. (J. Label Compd. Radiopharm. 2009, 52, 327-330) produced [18F]SFB via 4-[18F]fluorobenzaldehyde ([18F]FB-CHO) (Scheme 2). To a solution of K2CO3 and K2.2.2 in aqueous MeCN irradiated target water was added. The solvent was removed under reduced pressure in a flow of nitrogen and the residue was three times azeotropically dried by using MeCN. The residue was taken up in a solution of N,N,N-trimethyl-4-formylanilinium triflate in DMSO or DMF. The reaction mixture was briefly heated using microwave energy. The mixture was transferred on a silica gel cartridge and most of the solvent was removed by flushing with nitrogen. The intermediate [18F]FB-CHO was fractionally eluted using anhydrous ethyl acetate. One fraction (0.5 mL) was cooled to 0 C. Iodobenzene diacetate (BAIB) was added, the mixture was stirred at 0° C. for 15 min and allowed to reach ambient temperature for 5 min. It was decanted and the supernatant was purified via SPE or HPLC to afford [18F]SFB in EtOAc/hexane solution. Before [18F]SFB can be used for labelling of EtOAc/hexane-insoluble proteins or peptides, it should be taken up in an water-miscible solvent.
Additionally, the high hydrophobicity of the fluorobenzoyl group limits its application for the labelling of small molecules and shorter peptides. Therefore, several alternatives to SFB were proposed. The most interesting one is 6-[18F]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester ([18F]F-Py-TFP) first published by Olberg et al. (J. Med. Chem. 2010, 53, 1732-1740). [18F]F-Py-TFP could be prepared using a one-step procedure in moderate radiochemical yield (RCYs) of 40-50% (Scheme 3). Additionally, [18F]F-Py-TFP is more hydrolytically stable compared to [18F]SFB. Moreover, it provides more hydrophilic radiolabelled conjugates.

According to Olberg et al. 18F− was fixed on an anion exchange resin. Thereafter, it was eluted with a solution of tetrabutylammonium bicarbonate in 50% MeCN. The solvent was removed under reduced pressure in a flow of nitrogen and the residue was two times azeotropically dried by using MeCN. After that, a solution of the respective precursor, N,N,N-trimethyl-5-[(2,3,5,6-tetrafluorophenoxy)-carbonyl]pyridine-2-aminium trifluoromethanesulfonate 4c, in MeCN/tBuOH 2:8 was added to the [18F]TBAF and the reaction mixture was heated at 40 C for 10 min to give [18F]F-Py-TFP 3c in 50% RCY. [18F]F-Py-TFP was purified by SPE on a mixed mode reversed phase cation exchange resin. This synthetic method includes azeotropic drying steps. These preparation steps cause longer synthesis time and this results in lower RCYs of [18F]F-Py-TFP 3c. Furthermore, formation of [18F]F-Py-TFP is accompanied by the concurrent formation of significant amounts of 2,3,5,6-tetrafluorophenyl 6-(2,3,5,6-tetrafluorophenoxy)nicotinate which should be completely separated from the radiolabelled active ester best by HPLC.
In recent years imaging of prostate carcinoma (PCa) with PET isotope labelled PSMA ligands has become of considerable importance in clinical diagnosis. This can be mainly attributed to the high expression of the extracellular localized prostate specific membrane antigen (PSMA) in PCa. Ligands bearing the syL-C(O)-Glu-binding motif exhibit high binding affinity to PSMA. Pomper et al. (WO2010/01493; Clin. Cancer Res. 2011, 17, 7645-7653) exploited this lead structure to prepare 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid ([18F]DCFPyL) (Scheme 4). This PET tracer provided a clear delineation of PSMA positive prostate tumor xenografts in mice with an excellent tumor to background ratio.

Pomper et al. prepared [18F]DCFPyL in three steps. First, [18F]F-Py-TFP was synthesized as reported by Olberg et al. (J. Med. Chem. 2010, 53, 1732-1740). [18F]F-Py-TFP was eluted from the resin with dichloromethane into a vial containing 1,5-bis(4-methoxyphenyl)methyl 2-[({6-amino-1-[(4-methoxyphenyl)methoxy]-1-oxohexan-2-yl}carbamoyl)amino]pentanedioate [H-DUPA(OPMB)3] and triethylamine 11 (Scheme 4). The reaction mixture was heated at 45° C. for 20 min. Thereafter the solvent was removed with a stream of nitrogen. Anisole in TFA was added, the reaction mixture was heated at 45° C. for further 10 min and the desired product was isolated via HPLC using 10% MeCN (0.1% TFA). The fraction containing [18F]DCFPyL was neutralized with sodium bicarbonate, concentrated to dryness under reduced pressure and reconstituted in PBS to give [18F]DCFPyL in RCYs of 50-65%. The disadvantages of the above-mentioned method are:                Application of dichloromethane as a solvent        Two evaporation steps        PMB deprotection step using toxic TFA (Scheme 4B step 3)        Neutralisation step        Formulation step        
It is the objective of the present invention to provide an inventive method for preparation of PSMA selective PET tracer for prostate tumor imaging comprising a simplified procedure for the fast and high yielding preparation of 18F-labelled active esters and a pharmaceutical composition containing at least one compound (I) prepared by the inventive method for use in positron emission tomography (PET) imaging, especially imaging prostate tumor.
The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application.